Bacillus inaquosimilis strain and application thereof in ramie degumming

Abstract

The present application relates to a strain of Inhernia Bacillus and its application in ramie degumming, the strain is preserved in Guangdong Microbial Culture Collection Center on September 15, 2025, and the preservation number is GDMCC NO: 66966. When the strain is applied to ramie degumming, the degumming reaction can be carried out at a higher temperature, the degumming efficiency of ramie is effectively improved, the fiber is not damaged, the quality of the fiber is improved, and the strain has high application value in ramie degumming.

Description

Technical Field

[0001] This invention relates to the field of microbial degumming technology for ramie, and more specifically, to a strain of Bacillus subtilis and its application in ramie degumming. Background Technology

[0002] The fibers of bast fibers in ramie (Boehmeria nivea L.) and other bast fibers are largely coated or embedded with bonded non-cellulose gum components, which are difficult to remove using simple physical processing methods. Chemical degumming methods, with caustic soda boiling as their core technology, severely pollute the environment. Modern biological degumming technology represents the future direction of bast fiber processing, mainly divided into enzymatic degumming and microbial degumming. In actual production, enzymatic degumming is relatively expensive, while microbial degumming technology is more mature and practical. Currently, large-scale and stable application of biological degumming technology in production is still rare, mainly due to the lack of strains with strong degumming ability and stable properties. The foundation of microbial degumming is obtaining highly efficient strains; therefore, screening for superior degumming microorganisms has become crucial for the promotion of biological degumming technology in bast fibers.

[0003] Thermophilic microorganisms can grow and reproduce in high-temperature environments, and the enzymes they produce have good heat resistance, maintaining good thermal stability during production and transportation. They have high application value in ramie degumming. However, currently, there are no reports on the application of thermophilic microorganisms in ramie degumming. Summary of the Invention

[0004] In view of the above-mentioned technical problems existing in the prior art, the present invention provides a strain of Bacillus spp. that can grow in a high-temperature environment and the enzymes produced by it have good heat resistance and can maintain good thermal stability during transportation.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A strain of Bacillus haynesii was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO: 66966.

[0007] The present invention also provides the application of the above-mentioned strains in ramie degumming.

[0008] In some embodiments, the application method is as follows: the *Bacillus* strain is inoculated into a fermentation medium and fermented at a temperature of 40-50°C to obtain a fermentation broth; ramie bast fibers are mixed with the fermentation broth to carry out a degumming reaction.

[0009] In some embodiments, the inoculation amount of the strain is 2%-10%.

[0010] In some embodiments, the fermentation medium is a nutrient broth medium.

[0011] In some embodiments, the fermentation medium comprises 2.0-3.5g beef extract, 8-15g peptone, 3-8g sodium chloride, and 1000mL distilled water; more specifically, the fermentation medium comprises 3g beef extract, 10g peptone, 5g sodium chloride, and 1000mL distilled water. If a solid fermentation medium is used, 15-20g agar is added.

[0012] The present invention also provides a microbial inoculant, which includes the above-mentioned Bacillus spp. strain and / or its fermentation broth; the fermentation broth is obtained by inoculating the strain into a nutrient broth medium and fermenting it at 40-50°C.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] This invention provides a *Bacillus* strain that can grow at relatively high temperatures (45-60℃) and maintains good thermal stability during production and transportation. Furthermore, it can simultaneously secrete xylanase, pectinase, and mannanase, but does not produce cellulase, and the enzymes it produces have good heat resistance. When applied to ramie degumming, it allows the degumming reaction to occur at higher temperatures, effectively improving degumming efficiency without damaging the fiber, thereby improving fiber quality. This invention has high application value in ramie degumming and provides a new pathway for research on microbial degumming of ramie. Attached Figure Description

[0015] Figure 1 This is the phylogenetic tree of strain PQ-1;

[0016] Figure 2 Microscopic morphology of strain PQ-1;

[0017] Figure 3 This is a staining image of the spores of strain PQ-1;

[0018] Figure 4 The growth status of strain PQ-1 at different temperatures;

[0019] Figure 5 The fiber morphology of ramie before and after degumming is shown. Samples 1, 2, and 3 are ramie fibers after degumming by strain PQ-1, while the control group is raw ramie fiber in distilled water. Detailed Implementation

[0020] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] A domestic Bacillus strain, named PQ-1, was deposited on September 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO:66966.

[0023] To further understand the present invention, the technical solution of the present invention will now be described in detail with reference to preferred embodiments.

[0024] The reagents and culture media used in the following examples are as follows:

[0025] Screening medium: 1g ramie bast powder, 0.5g K2HPO4, 0.5g NaH2PO4, 2g (NH4)2SO4, 0.2g MgSO4·7H2O, 0.1g CaCl2, diluted to 1000mL with distilled water, sterilized at 121℃ for 20min. For solid screening medium, an additional 20g of agar powder should be added.

[0026] Liquid nutrient broth culture medium: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, diluted to 1000mL with distilled water, sterilized at 121℃ for 20min.

[0027] Solid nutrient broth medium (ordinary nutrient agar medium): Add 20g of agar powder to the liquid broth medium.

[0028] Induction medium: Add 4% ramie to the nutrient broth medium and sterilize at 121℃ for 20 min.

[0029] Pectinase-inducing medium: Add 1% pectin to the ordinary nutrient agar medium.

[0030] 1% Carboxymethyl cellulose sodium (CMC-Na) buffer: Dissolve 0.5 g of CMC-Na in 50 mL of pH 5.8 phosphate buffer;

[0031] 1% xylan buffer: Dissolve 0.5g xylan in 50ml pH 5.8 phosphate buffer;

[0032] 1% Pectin Buffer: Dissolve 0.5g of pectin in 50ml of pH 5.8 phosphate buffer;

[0033] 1% Mannan Buffer: Dissolve 0.5g of mannan in 50ml of pH 5.8 phosphate buffer;

[0034] DNS solution preparation: Dissolve 18.2g of potassium sodium tartrate in 50ml of distilled water, heat, and then add 0.63g of 3,5-dinitrosalicylic acid, 2.1g of NaOH, 0.5g of phenol and 0.5g of sodium sulfite to the hot solution in sequence. Stir until dissolved, cool, and then dilute to 100ml with distilled water. Store in a brown bottle at room temperature.

[0035] Peptone and beef extract were purchased from Beijing Aoboxing Biotechnology Co., Ltd.; potassium sodium tartrate and phenol were purchased from Tianjin Yongda Chemical Reagent Co., Ltd.; xylose, glucose, and 3,5-dinitrosalicylic acid were purchased from Sinopharm Group; pectin, mannan, sodium carboxymethyl cellulose, and xylan were purchased from Nanjing Songguan Biotechnology Co., Ltd.; and raw ramie was donated by the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences.

[0036] Example 1: Enrichment, Isolation, and Screening of Strains

[0037] The inventors collected hot spring water samples with silt from the hot springs in Wentang Town, Yichun.

[0038] Add 8g of raw ramie to 100mL of water sample and incubate at 55℃ and 180r / min for 24h to obtain the culture solution.

[0039] Take 10 mL of the above culture medium and inoculate it into 100 mL of sterile distilled water containing 4 g of raw ramie hemp. Incubate at 55 °C and 180 r / min for 24 h.

[0040] Repeat the above transfer process until the ramie surface is soft and the fibers are dispersed (i.e., degummed). Stop the transfer, and the final degummed liquid is the enriched bacterial solution.

[0041] Take 0.1 mL of the above-mentioned enriched bacterial solution, serially dilute it with sterile water, spread it on a solid selection medium plate, and incubate it at 45℃ for 24 h; pick colonies with different morphologies and transparent zones from the selection medium plate, streak them onto ordinary nutrient agar plates for purification, and incubate them at 45℃ for 24 h to obtain 30 strains.

[0042] The colonies of the 30 strains were numbered, and then single colonies of each strain were inoculated into induction medium. After culturing at 55℃ and 180r / min for 24h, the supernatant was collected by centrifugation to obtain crude enzyme solution.

[0043] The activity of cellulase in the crude enzyme solution was determined.

[0044] Pectinase is a key enzyme in ramie degumming. Strains lacking cellulase activity were screened and inoculated onto pectinase induction medium to screen for pectinase-producing strains. The selected pectinase-producing strains were then inoculated again into the induction medium, and crude enzyme solutions were obtained after cultivation. The activities of pectinase, xylanase, and mannanase were then measured.

[0045] The activities of cellulase, hemicellulase, pectinase, and mannanase in the crude enzyme solution were determined using the DNS method, as detailed below:

[0046] 1. Plot standard curves for glucose, xylose, mannose, and galacturonic acid respectively to calculate the enzyme activities of cellulase, xylanase, mannanase, and pectinase:

[0047] Construction of the glucose standard curve: Prepare a 1 mg / mL glucose stock solution by dissolving glucose in distilled water. Take 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 mL of the stock solution into clean colorimetric tubes, and bring the volume to 2 mL with ultrapure water. Add 2.5 mL of DNS solution and shake well. Incubate in a boiling water bath for 5 min, rapidly cool, and then bring the volume to 10 mL with ultrapure water. Mix well. Measure the OD value of 100 μL at 540 nm using a microplate reader. Plot the standard curve with glucose concentration (mg) on ​​the x-axis and the OD value measured at 540 nm on the y-axis. The equation of the curve is y = 0.6101x - 0.0092, R0. 2 =0.9987;

[0048] The method for plotting the standard curve of xylose is the same as that for glucose; the equation for the standard curve of xylose is y = 0.1923x - 0.3333, R0 2 =0.9967;

[0049] The method for plotting the standard curve of mannose is the same as that for glucose; the equation for the standard curve of mannose is y = 0.304x - 0.0188, R0 2 =0.9992;

[0050] The method for constructing the standard curve of galacturonic acid is the same as that for glucose; the equation for the standard curve of galacturonic acid is y = 0.554x - 0.0268, R0. 2 =0.9997.

[0051] 2. Determination of enzyme activity

[0052] Take 1 mL of crude enzyme solution, dilute appropriately, and mix with 1 mL of 1.0% CMC-Na buffer, xylan buffer, pectin buffer, and mannan buffer, respectively. React in a 55℃ constant temperature water bath for 10 min. After removing from the water bath, add 2.5 mL of DNS reagent, boil in a boiling water bath for 5 min, cool under running water, and add water to a final volume of 10 mL. Measure the OD value at 540 nm and calculate the enzyme activity of each enzyme using the standard curves for each reducing sugar or acid. Use 1 mL of crude enzyme solution boiled for 5 min as a control. Enzyme activity unit (IU) is defined as the amount of enzyme required to produce 1 μmol of reducing sugar (or acid) per minute.

[0053] Enzyme activity (IU / mL) =

[0054] 10 3 1mg = 10 3 μg

[0055] absorbance value at A=540nm

[0056] b = intercept

[0057] N Supernatant dilution factor

[0058] K - Slope of the standard curve

[0059] t = reaction time of enzyme or crude enzyme solution (min)

[0060] m: Molecular weight of glucose, xylose, mannose, or galacturonic acid (g / mol)

[0061] Vs = Volume of crude enzyme solution added (mL)

[0062] The cellulase activities of each strain are shown in Table 1.

[0063] Table 1 Comparison of cellulase activities among strains

[0064] Colony numbering OD value of control group OD value of experimental group Enzyme activity (IU / mL) 1# 0.00 0.00 0 2# 0.00 0.00 0 3# 0.00 0.091 0.93 4# 0.00 0.00 0 5# 0.00 0.00 0 6# 0.00 0.190 1.84 7# 0.00 0.00 0 8# 0.00 0.001 0 9# 0.00 0.141 1.39 10# 0.00 0.627 5.88 11# 0.00 0.149 1.47 12# 0.00 0.122 1.213 13# 0.00 0.092 0.935 14# 0.00 0.792 7.41 15# 0.00 0.106 1.06 16# 0.00 0.136 1.34 17# 0.00 0.00 0 18# 0.00 0.00 0 19# 0.00 0.882 8.23 20# 0.00 1.138 10.45 21# 0.00 0.07 0.719 22# 0.00 0.222 2.11 23# 0.00 0 0 24# 0.00 0.146 1.411 25# 0.00 0.298 2.800 26# 0.00 0 0 27# 0.00 0 0 28# 0.00 0 0 29# 0.00 1.342 12.31 30# 0.00 0 0

[0065] As shown in Table 1, some strains possess cellulase activity. However, strains with high cellulase activity can damage ramie fibers during degumming. Furthermore, pectinase is a key enzyme in ramie degumming. Therefore, strains without cellulase activity were selected for further screening of pectin-producing enzyme strains and detection of the enzyme activities of xylanase, pectinase, and mannanase. The detection results are shown in Tables 2 and 3.

[0066] Table 2. Ratio of hydrolysis zone to colony diameter of pectinase-producing bacteria

[0067]

[0068] Table 3 Comparison of xylanase, pectinase and mannanase activities of each strain

[0069]

[0070] As shown in Tables 2 and 3, strain #5 has high activities of xylanase, pectinase and mannanase, indicating that it is a potential ramie degumming strain.

[0071] Example 2 Identification of the strain

[0072] Strain #5 underwent Gram staining and spore staining. Single colonies of strain #5 were sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of the strain was amplified using universal primers 27F and 1492R as upstream and downstream primers, and then sequenced. The obtained DNA sequence was submitted to the NCBI website and obtained accession number PX969692. The 16S rDNA was compared with that of other bacterial species, such as... Figure 1 The results showed that it was similar to Bacillus haynesii. Figure 2 As shown, its Gram staining is positive; as Figure 3 As shown, strain #5 contains spores.

[0073] This yielded a strain of Bacillus subtilis, which was named PQ-1 and deposited on September 15, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNO:66966.

[0074] Example 3: Optimal growth temperature of strain PQ-1

[0075] Strain PQ-1 was inoculated into nutrient broth at a 2% inoculum and cultured overnight for 12 hours at 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃ using a shaker at 180 rpm. The OD value of the fermentation broth was measured at 600 nm using a UV spectrophotometer. The results are as follows: Figure 4 As shown, 45℃ is its optimal growth temperature.

[0076] Example 4: Degumming effect of strain PQ-1 on ramie

[0077] Strains PQ-1 were inoculated at a 10% inoculum in 100 mL of distilled water containing 4 g of ramie bast fibers. Degumming was carried out in a shaker at 45℃ and 180 rpm for 48 h, with three replicates. The degummed ramie was filtered through a 300-mesh sieve and pounded three times with a wooden mallet (rinsing with water after each pounding). After pounding and rinsing, the degummed ramie was placed in a drying oven and dried at 50℃ to constant weight before weighing. The dried ramie was photographed to compare surface morphology. A control group was prepared by adding 4% ramie to the distilled water. Samples before and after the degumming reaction are shown below. Figure 5 As shown in Table 3, the weight of ramie fibers before and after the degumming reaction is shown in Table 3.

[0078] Table 4. Weight of ramie fibers before and after degumming

[0079]

[0080] From Table 4 and Figure 5 It is known that the strain PQ-1 isolated in this application can grow in a relatively high-temperature environment with ramie as the only carbon source, and has excellent degumming effect on ramie.

[0081] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of Bacillus spp. from the sea ( Bacillus haynesii The strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 15, 2025, with accession number GDMCC NO: 66966.

2. The application of the strain described in claim 1 in ramie degumming.

3. The application according to claim 2, characterized in that, The application method is as follows: the Bacillus spp. strain is inoculated into a fermentation medium and fermented at a temperature of 40-50℃ to obtain a fermentation broth; ramie bast fibers are mixed with the fermentation broth to carry out a degumming reaction.

4. The application according to claim 3, characterized in that, The inoculation amount of the strain is 2%-10%.

5. The application according to claim 3 or 4, characterized in that, The fermentation medium is a nutrient broth medium.

6. The application according to claim 5, characterized in that, The fermentation medium comprises 2.0-3.5g beef extract, 8-15g peptone, 3-8g sodium chloride, and 1000mL distilled water.

7. The application according to claim 6, characterized in that, The fermentation medium contains 3g beef extract, 10g peptone, 5g sodium chloride, and 1000mL distilled water.

8. A microbial inoculant, characterized in that, It includes the *Bacillus* strain of claim 1 and / or its fermentation broth; the fermentation broth is obtained by inoculating the strain into a nutrient broth medium and fermenting it at 40-50°C.

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